The secosteroid vitamin D plays a critical role in cell differentiation, proliferation and mineral homeostasis. As such, this hormone is essential for the development and maintenance of healthy bone. The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3, mediates its effects through the vitamin D receptor (VDR), a member of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors. The primary effect of vitamin D on mineral metabolism relates to its ability to promote calcium absorption in the kidney and small intestine. Recent studies have also revealed a direct role of vitamin D on osteoblastic bone formation, suggesting an anabolic role for VDR. Indeed, two VDR ligands, Calcitriol (1,25-(OH)2D3) and its synthetic analog Alfacalcidol (1alpha-hydroxyvitamin D3), have been approved for the treatment of osteoporosis. However, hypercalcemia that develops as a result of VDR-mediated intestinal calcium transport limits the widespread use of these drugs. For this reason, bone-promoting VDR modulators with reduced activity in the small intestine are highly desirable and remain a priority in VDR drug discovery programs. Traditionally, VDR modulators have been identified in an empirical manner with chemistry being guided primarily by in vitro ligand binding assays, followed by secondary transcriptional reporter gene assays that measure functional agonism/antagonism. These approaches have been successful in identifying new VDR ligands but none have demonstrated sufficient tissue selectivity to be approved for osteoporosis. The rational design of next generation tissue- selective VDR modulators will require more sophisticated screening methodologies. During this Phase 1 program, we will employ a novel strategy to tackle this important problem by using an approach that takes advantage of our current understanding of the molecular determinants of VDR action and the observation that tissue-selective NR pharmacology is mediated in large part by the ability of these transcription factors to interact in a differential manner with either transcriptional co-activators or co-repressors. At the core of this process is an optimized T7 phage display technology that is used to rapidly identify tissue-specific NR interacting proteins. These interacting proteins are then used to develop a "receptor conformation profiling tool" (RCPT) that will allow for the identification of novel compounds that enable differential cofactor interactions. The stated objective of this Phase I proposal therefore is to utilize an optimized T7 phage display technology to identify the full spectrum of VDR interacting proteins from multiple target tissues including the bone and small intestine. These VDR-protein interactions will then be confirmed in cells to determine their utility in the future development, during a Phase 2 funding period, of a cofactor- based RCPT aimed at identifying novel bone-promoting small molecule regulators of VDR that have reduced calcium absorbing activities in the small intestine. PUBLIC HEALTH RELEVANCE: Vitamin D, acting through the vitamin D receptor (VDR), is a hormone essential for the maintenance and development of healthy bone. Indeed, the active metabolite of vitamin D is approved by the FDA for the treatment of osteoporosis, a disease that results from the weakening of bones. The treatment of osteoporosis with vitamin D hormones is however limited by their major side effect of elevated blood calcium levels. This is due to VDR activity in the small intestine, where vitamin D promotes intestinal calcium absorption. Our proposed goal is to use a state-of-art protein-protein interaction screening technology to develop and validate a novel drug discovery tool capable of identifying new VDR modulators that function differently than vitamin D. Successful completion of this Phase 1 project will allow, during a Phase 2 funding period, the implementation of this novel screening tool for use in the discovery of new classes of mechanistically distinct VDR modulators predicted to effectively treat osteoporosis with minimal side effects.